The Preparation Of Immunomagnetic Particles And Their Applications In Depleting Plasma High-Abundance Proteins

Characterization of low abundant proteins in serum has significant application for disease diagnosis, monitoring and drug therapy. However, high-abundance proteins, especially albumin tends to block the detection of low-abundance proteins. Depletion of high-abundance proteins is often adopted to enhance the sensitivity and resolution for detection of low abundance proteins. Due to their large surface area and superparamagnetism, magnetic particles are very promising in the application of protein separation, up to now, there is few reports on the research of immunodepleting plasma high-abundance proteins using magnetic particles as antibody carriers.The purpose of this study is to establish a method for removing albumin using immunomagnetic particles as carriers. Two kinds of magnetic particles (GoldMag particles and the functional magnetic particles terminated by 1,4-phenylene diisothiocyanate group) were selected as carriers for coupling anti-albumin polyclonal antibody and the method for depleting albumin from plasma was optimized. The specificity and reproducibility of this depletion method was evaluated by UV absorption detection, electrophoresis and Western blot. The results showed that the factors affecting the coupling amount are mainly pH value, type of coupling buffer and antibody concentration. The impact of coupling time was not significant. The optimal coupling condition is 0.02 M Tris-HCl buffer and 1.5 mg/mL antibody under pH 8.0 with 30 min reaction time. Under the optimized conditions,0.22 mg antibody was coupled on one milligram of the functional magnetic particles. Immunomagnetic particles can effectively remove the albumin in plasma, with the capability of 1.3μg plasma per mg immunomagnetic particle. And the results showed specificity and reproducibility. The entire removal process is easy, taking only 50 min.The yolk antibody has been proved to have many advantages in the immunology areas. Herein, three IgY antibodies with high titer and high purity (including anti-albumin, anti-IgG, anti-transferrin) were produced and the yolk antibody-based immunomagnetic particles were prepared. Average 0.31 mg of antibody IgY can be coupled on one milligram of functional magnetic particles. The immunomagnetic particles were used for a single component removal of albumin, IgG, transferrin, and multi-component removal of all the three high-abundance proteins. By UV absorption and electrophoresis, we evaluated the effect of yolk antibody-based immunomagnetic particles for single component protein removal, as well as multi-component protein. A protocol adopting yolk antibody-based immunomagnetic particles for removal of high abundance proteins in human plasma was set up. One mg of Yolk antibody-based immunomagnetic particles can effectively remove with the ability of the albumin from 2.8μg plasma, the IgG from 3.7μg plasma and the transferrin from 5.3μg plasma. The specificity and reproducibility were also tested. Mixed immunomagnetic particles can effectively remove of multi-components proteins at one time.